Cell Suspension Preparation

1. Hypotonic treatment
   Hypotonic treatment is critical for chromosome spreading. Too long or too short hypotonic incubations lead to inadequate spreading and poor banding or hybridization quality. For peripheral blood lymphocytes hypotonic treatment, it is recommended to incubate the cells in 0.075 M KCl at room temperature for 15 minutes. 
2. Cell washing
   After hypotonic incubation, cell suspensions should be washed several times in fresh fixative solution (methanol : acetic acid = 3:1). To make the washing easier and faster, the cells can be transferred to 1.5 microfuge vials. After washing centrifuge the cells at 6000 rpm for 1-2 minutes to collect the cells. 
3. Storage
   Cell suspensions can be stored in 1.5 ml or 2.0 ml microfuge vials filled with fixative at -20℃ for several years.

1. Slide treatment
   Commercially pre-cleaned slides do not require any extra cleaning steps prior to use. If the slides were not pre-cleaned, clean the slides by soaking the slide/coverslip in acetone, HCl, acetic acid and water prior to use. 
2. Volume of cell dropping
   10-20 ul cell suspension can be dropped on a slide by using a 200 ul pipette. 
3. Dropping from a height
   It is not necessary. 
4. Methods to control the degree of spreading
   The chromosome spreading takes place at the time when the fixative surface, as it evaporates, comes in contact with the spherical cell surfaces. This is the critical moment to intervene in order to increase or decrease the degree of spreading. Water vapors, heat or variable acetic acid concentrations can be used. 
5. How to reduce the influence of the atmospheric humidity
   After dropping cell suspension on the slide, the fixative evaporates. When the surface of the slide becomes grainy, put the slide face-down in the steam of a hot waterbath for 1-3 seconds, and then let the slide dry at room temperature. The hot steam will reduce the influence of the atmospheric humidity. 
6. Slide aging
   The slides should be aged at 37°C for 1-2 day before use. Over-aged metaphase spread may result weak and unspecific signals. It is not recommended to use slides aged at 37°C longer than 1 week.

1. Slide denaturing
   Metaphase spread slides should be denatured in denaturing solution for 5 minutes at 75℃ followed by dehydrting in 70%, 85% and 100% ethanol 1 min each. Over-aged slides need more time for denaturing. 
2. Probe denaturing
   The probe should be denatured 5 minutes at 75℃ followed by cooling at room temperature. Probe pre-annealing is not essential. 
3. Hybridization chamber
   To prevent the slides from drying during hybridization, place the slide in a moist hybridization chamber. Extra water below the slides does no harm but will be helpful to the hybridization when the coverslip sealing is incomplete.